Myosin-binding protein C (MyBP-C) is a component of the thick filaments of vertebrate skeletal and cardiac muscle. Phosphorylation of the cardiac isoform, cMyBP-C, plays a key role in modulating cardiac contractility in response to p-adrenergic stimulation. Mutations in cMyBP-C have been shown to be a prime cause of the cardiac disease, familial hypertrophic cardiomyopathy. Our long term goal is to understand the structural basis of cMyBP-C function. In this project electron microscopy and image processing will be used to elucidate the structure of the molecule, its organization in the sarcomere, its interaction with thin filaments, and the changes that occur when it is phosphorylated. Experiments will make use of expressed mutant and wild type cMyBP-C molecules and N-terminal fragments (produced in Core C), native thick filaments from wild type and transgenic hearts (Core C), and intact muscle. Three specific aims will be addressed. (1) How is cMyBPC organized at the molecular, thick filament and sarcomeric level? We will determine whether the MyBP-C molecule has specific structural features required to carry out its function, whether it wraps around, extends along, or projects away from the filament surface to interact with neighboring filaments, and how it influences myosin head organization. (2) What is the structural basis of cMyBP-C's modulation of actin-myosin interaction? We will determine whether cMyBP-C competes with myosin heads for actin binding, and whether it influences the position of tropomyosin on thin fliaments at high or low Ca[2+] levels. (3) What are the structural effects of cMyBP-C phosphorylation? We will determine whether phosphorylation alters cMyBP-C flexibility, thick filament structure (e.g. head conformation), and its interaction with thin filaments. These goals will be achieved using negative stain, cryo-EM, antibody labeling and muscle sectioning approaches, combined with single particle, helical and tomographic 3D reconstrucfion techniques. Results will be correlated with, and structurally underpin, parallel single molecule biophysics experiments (Project 2) and whole heart functional data (Project 3, Core B). The project will provide new insights into the structural mechanisms by which cMyBP-C funcfions in the heart.